1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns the detection of nucleic acids. In specific embodiments, the invention concerns methods and compositions for inhibiting the identification of a nucleic acid probe sequence used for the detection of a specific nucleic acid.
2. Description of Related Art
Nucleic acid sequences are made up of four distinct chemical bases: adenine, cytosine, guanine, and thymine (uracil in the case of RNA). Specific gene sequences consist of combinations of these four bases in a very specific order. Cellular DNA exists as a double-stranded heteroduplex in which adenine bases associate with thymine through hydrogen bonding and cytosine bases associate with guanine. Generally, nucleic acid diagnostic assays take advantage of these specific associations in multiple ways. For example, a detection oligonucleotide (probe) complementary to the specific DNA sequence being detected can be labeled with a variety of different detection molecules then the sample DNA can be immobilized and the presence or absence of the sequence of interest is determined by the binding and subsequent detection of the labeled probe. Alternatively, the probe can be immobilized onto a solid surface and used to capture the sample sequence of interest. Detection occurs through labeling of the sample DNA or the detection of the double-stranded sample/probe heteroduplex. In the case of real-time detection of a sequence the probe oligonucleotide is used during an amplification process of the sample sequence. The probe is often dual-labeled with a fluorescent molecule and a fluorescence quencher such that during the amplification process signal is generated through the release of the quenching molecule. Greater presence of sample results in greater amplification and thus greater and more rapid production of signal.
Sequence-specific nucleic acid hybridization assays are used for the detection of specific genetic sequences as indicators of genetic anomalies, mutations, and disease propensity. In addition, they are used for the detection of various biological agents and infectious pathogens. Because a complementary probe is required to detect the sequence of interest, nucleic acid sequencing techniques can rapidly determine the sequence of the probe being used in the assay and consequently the identity of the sequence of interest. The ability to reverse engineer a nucleic acid assay in this manner makes it easy to copy or circumvent the assay.
The ability to impede reverse engineering of nucleic acid hybridization assays would be highly valuable in a variety of circumstances. One example is protection from industrial espionage. Many of the sequences being used for medical diagnostic procedures as well as pathogen detection are part of the public domain; thus, it can be difficult for a company to maintain protection for the intellectual property produced from the utilization of specific sequences for diagnostic purposes. This is especially true in those foreign countries where little regard may be shown for any intellectual property protection. Thus, the only protection that companies have from industrial espionage through reverse engineering is keeping the specific sequences used for detection as a trade secret. Again, with the advances in nucleic acid technologies it is very easy and rapid to determine the sequences being used for nearly any detection platform, which allows a very rapid reproduction of specific assays.
Another situation in which it is desirable to impede reverse engineering of nucleic acid hybridization assays is the detection of agents for bioterrorism and biowarfare. In the case of bioterrorism and biowarfare, a primary concern is the presence of false positive samples (positive detection of the agent when the agent is not present) or false negatives (no detection of the agent when the agent is present).
A false positive sample, requires a similar involvement of first responders, medical staff, and a potential quarantine of the affected area as would be required for an actual attack. In the military a false positive can force troops to dress in biowarfare suits. Both responses cost money and diminish the availability and potency of the first responders and troops.
A false negative permits a real biological event to escape detection until significant symptomology occurs. This provides a chance for significant spread of an agent prior to detection and quarantine.
Because a false positive produces the same terror effect as a true positive sample and a false negative allows undetected spread of an agent, a detection platform must produce results with as much certainty as possible with as low as possible false positive and false negative results.
From a terrorist's perspective there are a number of ways to produce samples or agents that will result in a false positive or a false negative that directly relates to the detection system being used. Most nucleic acid detection platforms utilize a small unique DNA sequence to detect the presence or the absence of a gene that is indicative of a specific agent. However, if a terrorist became informed of the specific sequence that a detection platform uses for agent detection, a number of techniques could be used to produce false positive or false negative samples. Because the detection of an agent relies on the presence of that sequence but not necessarily the entire organism, introduction into a sample of a synthetic DNA sequence complementary to the nucleic acid probe being used for detection would produce the same result as if the entire organism was present.
In the same respect, to produce a false negative, the terrorist, knowing the detection sequence, could produce silent mutations in the organism that would change the detection sequence without changing the function of the gene product or the organism. Thus, the pathogenicity of the organism would not change, but the exquisite specificity of the detection platform would not indicate a positive sample since the detecting sequence in the organism had been changed.
Because of advances in nucleic acid technologies it is very easy and rapid to determine the sequences that are being used for nearly any detection platform; therefore, allowing false positive and false negative samples to be readily generated.
Clearly there is a need for methods to inhibit the reverse engineering of nucleic-acid based assays to protect intellectual property interest and to reduce the ability of a person to design a false-positive or false-negative agent to exploit a particular assay.